Apoptosis inhibitor

ABSTRACT

Disclosed is a method for treatment of a subject having a disease or condition associated with apoptosis, which comprises administering to the subject an effective amount of a 15-keto-prostaglandin compound represented by the following formula (I):

TECHNICAL FIELD

[0001] The present invention relates to a new use of 15-ketoprostaglandin compound as an apoptosis inhibitor.

BACKGROUND OF THE INVENTION

[0002] Apoptosis is a kind of genetically programmed cell death.Morphologically, apoptosis of a cell occurs along with the process asfollows: condensation of the nucleus of the cell; cell shrinkage;cytoplasmic vacuolation and cell surface smoothing; enlargement ofintercellular space; release of the cell from the pericellular region;fragmentation of the cell (to provide apoptosis body) and phagocytosisof the fragment by macrophage or the like. Biochemically, nucleosomalDNA is cleaved by endonuclease into 180-220 bp DNA fragments (ImmunologyToday 7:115-119, 1986; Science 245:301-305, 1989, the cited referencesare herein incorporated by reference.)

[0003] It has been revealed that apoptosis plays a role not only inphysiological cell death concerning generation/differentiation and turnover of normal tissues and cells, but also in some conditions ordiseases such as nerve cells death by ischemia after cerebralinfarction, cell death by radioisotope or anti cancer agent, cell deathby a toxin or virus infection, lymphocytopenia due to virus infectionsuch as AIDS, autoimmune disease, Alzheimer disease and inflammatory.Further, apoptosis plays a role in photoreceptor cell death observed inlight induced retinal photic injury (Current Eye Research Vol. 10 No.1:47-59, 1991, the cited reference is herein incorporated by reference).Accordingly, development of new apoptosis controlling drugs (that is,apoptosis inhibitor and apoptosis inducer) are expected to provide newtype of drugs with novel mode of action useful in a variety of fieldssuch as immune system, cerebral nerve system, optic nerve system,cancer, aging and the like.

[0004] Prostaglandins (hereinafter, referred to as PG(s)) are members ofclass of organic carboxylic acids, which are contained in tissues ororgans of human or most other animals, and exhibit a wide range ofphysiological activity. PGs found in nature (primary PGs) generally havea prostanoic acid skeleton as shown in the formula (A):

[0005] On the other hand, some of synthetic analogues of primary PGshave modified skeletons. The primary PGs are classified to PGAs, PGBs,PGCs, PGDS, PGEs, PGFs, PGGs, PGHs, PGIs and PGJs according to thestructure of the five-membered ring moiety, and further classified intothe following three types by the number and position of the unsaturatedbond at the carbon chain moiety:

[0006] subscript 1: 13,14-unsaturated-15-OH

[0007] subscript 2: 5,6- and 13,14-diunsaturated-15-OH

[0008] subscript 3: 5,6-, 13,14- and 17,18-triunsaturated-15-OH.

[0009] Further, the PGFs are classified, according to the configurationof the hydroxy group at position 9, into a type (the hydroxy group is ofa α-configuration) and β type (the hydroxy group is of aβ-configuration).

[0010] PGE₁, PGE₂ and PGE₃ are known to have vasodilation, hypotension,gastric secretion decreasing, intestinal tract movement enhancement,uterine contraction, diuretic, bronchodilation and anti ulceractivities. PGF_(1α), PGF_(2α) and PGF_(3α) have been known to havehypertension, vasoconstriction, intestinal tract movement enhancement,uterine contraction, lutein body atrophy and bronchoconstrictionactivities.

[0011] In addition, some 15-keto prostaglandins (i.e. those having anoxo group at position 15 in place of the hydroxy group) and13,14-dihydro-15-keto-prostaglandins are known as substances naturallyproduced by enzymatic actions during in vivo metabolism of primary PGs.15-keto PGs have been disclosed in the specification of U.S. Pat. Nos.5,073,569, 5,166,174, 5,221,763, 5,212,324 and 5,739,161. (These citedreferences are herein incorporated by reference.)

[0012] As apoptosis inhibitors, Interleukine-1 converting enzymeinhibitor and basic fibroblast growth factor (bFGF) have been known.Further, isocarbacycline derivative inhibits apoptosis of nerve cells(European patent application Laid Open No. 911314, the cited referenceis herein incorporated by reference), and prostaglandin E₁ inhibitsdaunorbicin-induced apoptosis of human leukaemic cells. (JapaneseJournal of Inflammation Vol. 18, No. 5:369-376, 1988; the citedreference is herein incorporated by reference).

SUMMARY OF THE INVENTION

[0013] An object of the present invention is to provide an apoptosisinhibitor, which is useful for treatment of various conditions anddiseases associated with apoptosis.

[0014] The inventors have studied on bioactivity of 15-ketoprostaglandin compounds and found that 15-keto-prostaglandin compoundsexpress a significant apoptosis inhibiting activity, and achieved to theinvention.

[0015] That is, the present invention provides an apoptosis inhibitingcomposition comprising a 15-keto-prostaglandin compound as an activeingredient.

[0016] Further, the present invention provides a method for treatment ofa subject having a disease or condition associated with apoptosis whichcomprises administering an effective amount of a 15-keto-prostaglandincompound to the subject.

[0017] Further more, the present invention provides use of a15-keto-prostaglandin compound for producing a pharmaceuticalcomposition for treatment of a subject having a disease or conditionassociated with apoptosis.

[0018] In the present invention, the “15-keto-prostaglandin compounds”(hereinafter, referred to as “15-keto-PG compounds”) may include any ofderivatives or analogs (including substituted derivatives) of a compoundhaving an oxo group at 15-position of the prostanoic acid skeletoninstead of the hydroxy group, irrespective of the configuration of thefive membered ring, the number of double bonds, presence or absence of asubstituent, or any other modification in the α or ω chain.

[0019] The nomenclature of the 15-keto-PG compounds used herein is basedon the numbering system of prostanoic acid skeleton represented in theabove formula (A).

[0020] The formula (A) shows a basic skeleton of the C-20 carbon atoms,but the 15-keto-PG compounds in the present invention are never limitedto those having the same number of carbon atoms. In the formula (A), thenumbering of the carbon atoms which constitute the basic skeleton of thePG compounds starts at the carboxylic acid (numbered 1), and carbonatoms in the α-chain are numbered 2 to 7 towards the five-membered ring,those in the ring are 8 to 12, and those in the ω-chain are 13 to 20.When the number of carbon atoms is decreased in the α-chain, the numberis deleted in the order starting from position 2; and when the number ofcarbon atoms is increased in the α-chain, compounds are named assubstitution compounds having respective substituents at position 2 inplace of the carboxy group (C-1). Similarly, when the number of carbonatoms is decreased in the ω-chain, the number is deleted in the orderstarting from position 20; and when the number of carbon atoms isincreased in the ω-chain, the carbon atoms beyond position 20 are namedas substituents. Stereochemistry of the compounds is the same as that ofthe above formula (A) unless otherwise specified.

[0021] In general, each of the terms PGD, PGE and PGF represents a PGcompound having hydroxy groups at positions 9 and/or 11, but in thepresent specification these terms also include those PG relatedcompounds having substituents other than the hydroxy group at positions9 and/or 11. Such compounds are referred to as9-dehydroxy-9-substituted-PG compounds or 11-dehydroxy-11-substituted-PGcompounds. A PG compound having hydrogen in place of the hydroxy groupis simply named as 9- or 11-dehydroxy compound.

[0022] As stated above, the nomenclature of 15-keto-PG compounds isbased on the prostanoic acid skeleton. However, in case the compound hasa similar partial construction as a prostaglandin, the abbreviation of“PG” may be used. Thus, a PG compound of which a chain is extended bytwo carbon atoms, that is, having 9 carbon atoms in the α chain isnominated as 2-decarboxy-2-(2-carboxyethyl)-15-keto PG compound.Similarly, a compound having 11 carbon atoms in the a chain is nominatedas 2-decarboxy-2-(4-carboxybutyl)-15-keto-PG compound. Further, a15-keto-PG compound of which ω-chain is extended by two carbon atoms,that is, having 10 carbon atoms in the ω-chain is nominated as15-keto-20-ethyl-PG compound. These compounds, however, may also benamed according to the IUPAC naming system.

DETAILED DESCRIPTION OF THE INVENTION

[0023] The 15-keto-PG compound used in the present invention may includeany PG derivative or analog insofar as of which C-15 constitute carbonylgroup, and may further include compounds having a 13,14-doublebond(15-keto-PG type 1 compound), 13-14 and 5-6 double bonds(15-keto-PGtype 2 compound), or 13-14, 5-6 and 17-18 double bonds (15-keto-PG type3 compound) as well as a 13,14-single bond (13,14-dihydro-15-keto-PGcompounds).

[0024] Typical examples of the compounds used in the present inventioninclude 15-keto-PG type 1, 15-keto-PG type 2, 15-keto-PG type 3,13,14-dihydro-15-keto-PG type 1, 13,14-dihydro-15-keto-PG type 2,13,14-dihydro-15-keto-PG type 3 and the derivatives thereof.

[0025] Examples of the substitution compounds or derivatives include a15-keto-PG compound of which carboxy group at the end of a chain isesterified; a compound of which a chain is extended; physiologicallyacceptable salt thereof; an unsaturated derivative having a double bondat 2-3 position or a triple bond at position 5-6, a PG compound havingsubstituent(s) at position(s) 3, 5, 6, 16, 17, 18, 19 and/or 20; and aPG compound having lower alkyl or a hydroxy (lower) alkyl group atposition 9 and/or 11 in place of the hydroxyl group.

[0026] According to the present invention, preferred substituents atpositions 3, 17, 18 and/or 19 include alkyl having 1-4 carbon atoms,especially methyl and ethyl. Preferred substituents at position 16include lower alkyl such as methyl and ethyl, hydroxy, halogen atomssuch as chlorine and fluorine, and aryloxy such astrifluoromethylphenoxy. Preferred substituents at position 17 includehalogen atoms such as chlorine and fluorine. Preferred substituents atposition 20 include saturated or unsaturated lower alkyl such as C₁₋₄alkyl lower alkoxy such as C₁₋₄ alkoxy, and lower alkoxy alkyl such asC₁₋₄ alkoxy-C₁₋₄ alkyl. Preferred substituents at position 5 includehalogen atoms such as chlorine and fluorine. Preferred substituents atposition 6 include an oxo group forming a carbonyl group.Stereochemistry of PGs having hydroxy, lower alkyl orhydroxy(lower)alkyl substituent at positions 9 and 11 may be α, β or amixture thereof.

[0027] Further, the above derivatives may be compounds having an alkoxy,cycloalkyl, cycloalkyloxy, phenoxy or phenyl group at the end of theω-chain where the chain is shorter than the primary PGs.

[0028] Especially preferred compounds include a 13,14-dihydro-15-keto-PGcompound which has a single bond at position 13-14; a 15-keto-16 mono ordi-halogen PG compound which has one or two halogen atoms such aschlorine and fluorine at position 16; a2-decarboxy-2-(2-carboxyethyl)-15-keto-PG compound in which skeletalcarbon of α chain is extended by two carbon atoms; and a 15-keto-PGEcompound which has an oxo group at position 9 and a hydroxyl group atposition 11 of the five memberd ring.

[0029] A preferred compound used in the present invention is representedby the formula (I):

[0030] wherein W₁, W₂ and W₃ are carbon or oxygen atoms;

[0031] L, M and N are hydrogen, hydroxy, halogen, lower alkyl, loweralkoxy, hydroxy(lower)alkyl or oxo, wherein at least one of L and M is agroup other than hydrogen, and the five-membered ring may have one ormore double bond(s);

[0032] A is —CH₂OH, —COCH₂OH, —COOH or its functional derivative,

[0033] B is —CH₂—CH₂—, —CH═CH— or —C≡C—;

[0034] R₁ is a divalent saturated or unsaturated lower-medium aliphatichydrocarbon residue, which is unsubstituted or substituted by halogen,alkyl, hydroxy, oxo, aryl or heterocyclic group; and

[0035] Ra is a saturated or unsaturated lower-medium aliphatichydrocarbon residue, which is unsubstituted or substituted by halogen,oxo, hydroxy, lower alkyl, lower alkoxy, lower alkanoyloxy,cyclo(lower)alkyl, cyclo(lower)alkyloxy, aryl, aryloxy, heterocyclicgroup or heterocyclic-oxy group; cyclo(lower)alkyl;cyclo(lower)alkyloxy; aryl; aryloxy; heterocyclic group; orheterocyclic-oxy group.

[0036] A group of particularly preferable compounds among theabove-described compounds is represented by the general formula (II):

[0037] wherein L and M are hydrogen, hydroxy, halogen, lower alkyl,lower alkoxy, hydroxy(lower)alkyl or oxo, wherein at least one of L andM is a group other than hydrogen, and the five-membered ring may haveone or more double bond;

[0038] A is —CH₂OH, —COCH₂OH, —COOH or its functional derivative;

[0039] B is —CH₂—CH₂—, —CH═CH— or —C≡C—;

[0040] X₁ and X₂ are hydrogen, lower alkyl or halogen;

[0041] R₁ is a divalent saturated or unsaturated lower-medium aliphatichydrocarbon residue, which is unsubstituted or substituted by halogen,alkyl, hydroxy oxo, aryl or heterocyclic group;

[0042] R₂ is a single bond or lower alkylene; and

[0043] R₃ is lower alkyl, lower alkoxy, cyclo(lower)alkyl,cyclo(lower)alkyloxy, aryl, aryloxy, heterocyclic group orheterocyclic-oxy group.

[0044] In the above formulae, the term “unsaturated” in the definitionsfor R₁ and R_(a) is intended to include one or more double bonds and/ortriple bonds that are isolatedly, separately or serially present betweencarbon atoms of the main and/or side chains. An unsaturated bond betweentwo serial positions is represented by denoting the lower number of thetwo positions, and an unsaturated bond between two distal positions isrepresented by denoting both of the positions. Preferred unsaturatedbonds are a double bond at position 2 and a double or triple bond atposition 5.

[0045] The term “lower-medium aliphatic hydrocarbon” means a hydrocarbonhaving a straight or branched chain of 1 to 14 carbon atoms, wherein theside chain has preferably 1 to 3 carbon atoms. The preferred R_(a) has 1to 10, more preferably 6 to 10 carbon atoms, and the preferred R_(a) has1 to 10, more preferably 1 to 8 carbon atoms.

[0046] The term “halogen” includes fluorine, chlorine, bromine andiodine.

[0047] The term “lower” means a group having 1 to 6 carbon atoms unlessotherwise specified.

[0048] The term “lower alkyl” means a straight- or branched-chainsaturated hydrocarbon group having 1 to 6 carbon atoms, for example,methyl, ethyl, propyl, isopropyl, butyl, isobutyl, t-butyl, pentyl andhexyl.

[0049] The term “lower alkoxy” means a lower alkyl-O— wherein the loweralkyl is as defined above.

[0050] The term “hydroxy(lower)alkyl” means a lower alkyl as definedabove, which is substituted by at least one hydroxyl group, for example,hydroxymethyl, 1-hydroxyethyl, 2-hydroxyethyl and1-methyl-1-hydroxyethyl.

[0051] The term “lower alkanoyloxy” means a group represented by theformula RCO—O—, wherein RCO— is an acyl formed by oxidation of a loweralkyl as defined above, for example, acetyl.

[0052] The term “cyclo(lower)alkyl” means a group formed by cyclizationof a lower alkyl group as defined above but contains 3 or more carbonatoms, for example, cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl.

[0053] The term “cyclo(lower)alkyloxy” means a group represented by theformula cyclo(lower)alkyl-O—, wherein cyclo(lower)alkyl is as definedabove.

[0054] The term “aryl” includes optionally substituted aromatichydrocarbon ring, preferably monocyclic group, for example, phenyl,naphthyl, tolyl and xylyl. Examples of the substituents include halogen,lower alkoxy and halo(lower) alkyl group, wherein halogen atom and loweralkyl group are as defined above.

[0055] The term “aryloxy” means a group represented by the formula ArO—,wherein Ar is an aryl group as defined above.

[0056] The term “heterocyclic group” includes mono- to tri-cyclic,preferably monocyclic heterocyclic group which is 5 to 14, preferably 5to 10 membered ring having optionally substituted carbon atom and 1 to4, preferably 1 to 3 of 1 or 2 kinds of hetero atoms selected fromnitrogen atom, oxygen atom and sulfur atom. Examples of the heterocyclicgroup include furyl, thienyl, pyrrolyl, oxazolyl, isoxazolyl, thiazolyl,isothiazolyl, imidazolyl, pyrazolyl, furazanyl, pyranyl, pyridyl,pyridazyl, pyrimidinyl, pyrazyl, 2-pyrrolinyl, pyrrolidinyl,2-imidazolinyl, imidazolidinyl, 2-pyrazolinyl, pyrazolidinyl,piperidino, piperazinyl, morpholino, indolyl, benzothienyl, quinolyl,isoquinolyl, puryl, quinazolinyl, carbazolyl, acridinyl,phenanthridinyl, benzimidazolyl, benzimidazolonyl, benzothiazolyl andphenothiazinyl. Examples of the substituent in this case include halogenand halogen substituted lower alkyl, wherein halogen atom and loweralkyl are as defined above.

[0057] The term “heterocyclic-oxy group” means a group represented bythe formula HcO—, wherein Hc is a heterocyclic group as defined above.

[0058] The term “functional derivative” of A includes salts (preferablypharmaceutically acceptable salts), ethers, esters and amides.

[0059] Examples of suitable “pharmaceutically acceptable salts” includecommonly used nontoxic salts such as salts with inorganic bases, forexample, alkali metal salts (sodium salt, potassium salt and the like);alkaline earth metal salts (calcium salt, magnesium salt and the like);ammonium salts; salts with organic bases, for example, amine salts (suchas methylamine salt, dimethylamine salt, cyclohexylamine salt,benzylamine salt, piperidine salt, ethylenediamine salt, ethanolaminesalt, diethanolamine salt, triethanolamine salt,tris(hydroxymethylamino)ethane salt, monomethyl-monoethanolamine salt,procaine salt and caffeine salt); basic amino acid salts (such asarginine salt and lysine salt); tetraalkyl ammonium salts and the like.These salts may be manufactured from, for example, corresponding acidsand bases in accordance with a conventional manner or by the saltexchange process.

[0060] Examples of the ethers include alkyl ethers, for example, loweralkyl ethers such as methyl ether, ethyl ether, propyl ether, isopropylether, butyl ether, isobutyl ether, t-butyl ether, pentyl ether and1-cyclopropyl ethyl ether; medium or higher alkyl ethers such as octylether, diethylhexyl ether, lauryl ether and cetyl ether; unsaturatedethers such as oleyl ether and linolenyl ether; lower alkenyl etherssuch as vinyl ether and allyl ether; lower alkynyl ethers such asethynyl ether and propynyl ether; hydroxy(lower)alkyl ethers such ashydroxyethyl ether and hydroxyisopropyl ether; lower alkoxy (lower)alkylethers such as methoxymethyl ether and 1-methoxyethyl ether; optionallysubstituted aryl ethers such as phenyl ether, tosyl ether, t-butylphenylether, salicyl ether, 3,4-dimethoxyphenyl ether and benzamidophenylether; and aryl(lower)alkyl ethers such as benzyl ether, trityl etherand benzhydryl ether.

[0061] Examples of the esters include aliphatic esters, for example,lower alkyl esters such as methyl ester, ethyl ester, propyl ester,isopropyl ester, butyl ester, isobutyl ester, t-butyl ester, pentylester and 1-cyclopropylethyl ester; lower alkenyl esters such as vinylester and allyl ester; lower alkynyl esters such as ethynyl ester andpropynyl ester; hydroxy(lower)alkyl esters such as hydroxyethyl ester;and lower alkoxy(lower)alkyl esters such as methoxymethyl ester and1-methoxyethyl ester, and as well as, for example, optionallysubstituted aryl esters such as phenyl ester, tolyl ester, t-butylphenylester, salicyl ester, 3,4-dimethoxyphenyl ester and benzamidephenylester; and aryl(lower)alkyl esters such as benzyl ester, trityl esterand benzhydryl ester. Examples of amides include mono- or di-lower alkylamides such as methylamide, ethylamide and dimethylamide; aryl amidessuch as anilide and toluidide; and alkyl or aryl sulfonyl amides such asmethylsulfonyl amide, ethylsulfonyl amide and tolylsulfonyl amide.

[0062] Preferred examples of L and M include hydroxy and oxo andespecially, M is hydroxy and L is oxo which provides the 5-membered ringstructure of, so called, PGE type.

[0063] Preferred examples of A-group include —COOH and itspharmaceutically acceptable salts, esters and amides.

[0064] Preferred example of B is —CH₂—CH₂— which provides the structureof so-called, 13,14-dihydro type.

[0065] Preferred example of X₁ and X₂ is that at least one of them ishalogen, more preferably, both of them are halogen, especially, fluorinethat provides a structure of, so called 16,16-difluoro type.

[0066] Preferred R₁ is a hydrocarbon containing 1-10 carbon atoms,preferably 6-10 and more preferably 8 carbon atoms.

[0067] Examples of R₁ include, for example, the following residues:

[0068] —CH₂—CH₂—

[0069] —CH₂—CH₂—CH₂—CH₂—

[0070] —CH₂—CH═CH—CH₂—,

[0071] —CH₂—C≡C—CH₂—,

[0072] —CH₂—CH₂—CH₂—CH₂—CH₂—,

[0073] —CH₂—CH═CH—CH₂—CH₂—

[0074] —CH₂—C≡C—CH₂—CH—

[0075] —CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—,

[0076] —CH₂—CH═CH—CH₂—CH₂—CH₂—,

[0077] —CH₂—CH₂—CH₂—CH₂—CH═CH—

[0078] —CH₂—C≡—C—CH₂—CH₂—CH₂—,

[0079] —CH₂—CH₂—CH₂—CH₂—CH(CH₃)—CH₂—

[0080] —CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—,

[0081] —CH₂—CH═CH—CH₂—CH₂—CH₂—CH₂—CH₂—,

[0082] —CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—CH═CH—,

[0083] —CH₂—C≡C—CH₂—CH₂—CH₂—CH₂—CH₂—,

[0084] —CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—CH(CH₃)—CH₂—,

[0085] Preferred Ra is a hydrocarbon containing 1-10 carbon atoms, morepreferably, 1-8 carbon atoms. Ra may have one or two side chains havingone carbon atom.

[0086] Preferred R₂ is a single bond or a saturated or unsaturatedbivalent lower to medium aliphatic hydrocarbon residue, which maypreferably have 1-10 carbon atoms, more preferably 1-8 carbon atoms,especially 1-6 alkylene.

[0087] Preferred R₃ is a hydrogen atom, aryl or aryloxy.

[0088] The configuration of the ring and the α- and/or ω chains in theabove formulae (I) and (II) may be the same as or different from thoseof the primary PGs. However, the present invention also includes amixture of a compound having a primary type configuration and a compoundof a non-primary type configuration.

[0089] Typical examples of the compounds used in the present inventioninclude 2-decarboxy-2-(carboxy lower alkyl)-15-keto-PG compounds,especially, 2-decarboxy-2-(2-carboxyethyl)-15-keto-PG compound and2-decarboxy-2-(4-carboxybutyl)-15-keto PG compound and 5-fluoro, 6-keto,11-dehydroxy, 16-fluoro, 16-methyl, 17-fluoro, 17-methyl, 18-methyl,19-methyl, 20-methyl, 20-ethyl, 20-propyl and 18,19,20-trinor-17-phenylderivatives thereof.

[0090] When a 15-keto-PG compound of the present invention has, forexample, a single bond between carbon atoms number 13 and 14, thecompound may be in the keto-hemiacetal equilibrium by formation of ahemiacetal between hydroxy group at position 11 and oxo at position 15.

[0091] If such tautomeric isomers as above are present, the proportionof both tautomeric isomers varies with the structure of the rest of themolecule or the kind of the substituent present. Sometimes one isomermay predominantly be present in comparison with the other. However, itis to be appreciated that the compounds used in the invention includeboth isomers. Further, while the compounds used in the invention may berepresented by a structure formula or name based on keto-type regardlessof the presence or absence of the isomers, it is to be noted that suchstructure or name does not intend to exclude the hemiacetal typecompound.

[0092] The present invention includes any of the isomers such as theindividual tautomeric isomers, a mixture thereof, or optical isomers, amixture thereof, a racemic mixture and other isomers such as stericisomers useful for the same purpose.

[0093] Other species compound useable in the present invention aredisclosed in U.S. Pat. Nos. 5,073,569, 5,166,174, 5,221,763, 5,212,324and 5,739,161 and U.S. patent application Ser. No. 09/011218 (thesecited references are herein incorporated by reference).

[0094] The active compounds used in the present invention may be usedfor treatment of animals and human beings having a condition associatedwith apoptosis. The compounds are usually applied systemically ortopically by such methods as ophthalmic instillation, oraladministration, intravenous injection (including infusion), subcutaneousinjection, intra rectal administration, intra vaginal administration andthe like. Especially, ophthalmic instillation is preferable. The dosagemay vary depending on the strain of the patient, i.e. particular animalor human, age, body weight, symptom to be treated, desired therapeuticeffect, administration route, term of treatment and the like. Asatisfactory effects may be obtained by topical administration of thecompound at the amount of 0.01-100 μg/eye, or by systemic administration2-4 times per day or continuous administration at the amount of0.001-500 mg/kg per day.

[0095] Examples of ophthalmic compositions of the present inventioninclude ophthalmic solution and ointment. The ophthalmic solution may beprepared by dissolving the active ingredient into sterilized aqueoussolution such as saline or buffer. A powder composition for ophthalmicsolution to be dissolved before use may also be used. The ophthalmicointment may be prepared by mixing the active ingredient with ointmentbase.

[0096] Examples of solid compositions for oral administration includetablets, troches, sublingual tablets, capsules, pills, powders, granulesand the like. The solid composition may be prepared by mixing one ormore active ingredients with at least one inactive diluent, e.g.lactose, mannitol, glucose, hydroxypropyl cellulose, fine crystallinecellulose, starch, polyvinyl pyrrolidone and magnesiumaluminometasilicate. The composition may further contain additives otherthan the inactive diluent, for example, lubricants e.g., magnesiumstearate, a disintegrator e.g. cellulose calcium gluconates, stabilizerse.g. α-, β- or γ-cyclodextrin, ether cyclodextrins, e.g. dimethyl-α-,dimethyl-β-, trimethyl-β- or hydroxypropyl-β-cyclodextrins, branchedcyclodextrins, e.g. glucosyl- or maltosyl-cyclodextrins, formylcyclodextrin, sulfur-containing cyclodextrin, misoprotol orphospholipids. When a cyclodextrin is used as a stabilizer, the activeingredient may form an inclusion compound with the cyclodextrin toimprove the stability. The stability may also be improved by includingthe ingredient in liposome made from phospholipid. Tablets and pills maybe coated with an enteric or gastroenteric film e.g. white sugar,gelatin, hydroxypropylcellulose, hydroxypropylmethyl cellulosephthalates and the like, if necessary. They may be covered with two ormore layers. Additionally, the composition may be in the form ofcapsules made from an easily degradable material such as gelatin.Sublingual tablet is preferable, when an immediate effect is desired.

[0097] Base of the composition may be glycerin, lactose and the like.Examples of liquid compositions for oral administration includeemulsions, solutions, suspensions, syrups, elixirs and the like. Saidcompositions may further contain a conventionally used inactive diluente.g. purified water or ethyl alcohol. The composition may containadditives other than the inactive diluent such as adjuvant e.g. wettingagents and suspending agents, sweeteners, flavors, fragrance andpreservatives.

[0098] The composition of the present invention may be in the form ofspray which contains one or more active ingredients and may be preparedaccording to a known method.

[0099] Examples of the injectable compositions of the present inventionfor parenteral administration include sterile aqueous or nonaqueoussolutions, suspensions and emulsions. Diluents for the aqueous solutionor suspension may include, for example, distilled water for injection,physiological saline and Ringer's solution.

[0100] Non-aqueous diluents for solution and suspension may include, forexample, propylene glycol, polyethylene glycol, vegetable oils such asolive oil, alcohols such as ethanol and polysorbate. The composition mayfurther comprise additives such as preservatives, wetting agents,emulsifying agents, dispersing agents and the like. These are sterilizedby filtration through, e.g. a bacteria-retaining filter, compoundingwith a sterilizer, or by means of gas or radioisotope irradiationsterilization. The injectable composition may also be provided as asterilized powder composition to be dissolved in a sterilized solventfor injection before use.

[0101] Another formulation of the composition according to the presentinvention may be rectal or vaginal suppository. Said suppository may beprepared by mixing at least one active compound according to theinvention with a suppository base e.g. cacao butter and may optionallybe admixed with a nonionic surfactant to improve absorption.

[0102] The term “treatment” used herein refers to any means of controlof a condition associated with apoptosis, including prevention, care,relief of the condition, and arrestation or relief of development of thecondition.

[0103] The apoptosis inhibiting composition of the present invention canbe applied for treatment of a various diseases and conditions associatedwith apoptosis. For example, the composition may be useful for treatmentof nerve cell death by ischemia after cerebral infarction or the like,malignant tumor, autoimmune disease such as lymphocytopenia caused byvirus infection such as AIDS, Alzheimer's disease, inflammation and eyedisorders caused by light irradiation such as photoretinitis.

[0104] The composition of the present invention may further be admixedwith any of pharmaceutically active agents in so far as said agent iscompatible with the purpose of the present invention.

EXAMPLE

[0105] The present invention will be illustrated in more detail by wayof the following examples. These examples should not be used as anylimitation of the present invention.

[0106] Test Example

[0107] (1 )Breeding Condition and Administration Method

[0108] SD strain rats (male, 11 weeks old) were continuously exposed to1000 lux of light for 4 days. During the exposure of light, the testgroup animals were administered subcutaneously with a compositioncomprising2-decarboxy-2-(2-carboxyethyl)-13,14-dihydro-15-keto-16,16-difluoro-20-ethyl-PGE,isopropyl ester of the following formula (IV) at the amount of 10 μg/kgof the active ingredient per single administration three times a day,for 4 days. The control group animals were administrated subcutaneouslythe same volume of the vehicle.

[0109] (2) Preparation and Staining

[0110] After the continuous light exposure was finished, animals of testand control groups were sacrificed by excessive etherization and botheyes of each animal were removed. The eyes were immediately fixed in a2% paraformaldehyde and 2.5% glutaraldehyde solution in phosphatebuffer, dehydrated with alcohol, and then embedded in paraffin. Thusfixed eyes were sliced parallel to the meridian of eye to provide thinretinal preparations each comprises optic disc. The obtained slices weresubjected to tunnel staining (Apoptag® Intergen Company).

[0111] (3) Estimation

[0112] Total cell number and the number of tunnel-positive cells per 200μm of the retina were counted and the ratio of the TUNEL-positive cellsto the total cell number was determined.

[0113] (4) Result

[0114] The ratio of the TUNEL-positive cells to the total cell number isshown in table 1. The less number of positive cells means the strongerapoptosis inhibition. TABLE 1 TUNEL-positive cell ratio Ratio of theTUNEL-positive cells (%) n (Ave ± SE) Control group 5 9.3 ± 0.8  TestGroup 5 1.5 ± 0.2**

[0115] The above result demonstrates the prostaglandin compound of thepresent invention has an apoptosis inhibiting activity.

INDUSTRIAL APPLICABILITY

[0116] The compound used in the present invention is useful as anapoptosis inhibitor. Therefore, said compound is expected to be usefulin treatment or prophylaxis of a various conditions and diseasesassociated with apoptosis.

What is claimed is
 1. A method for treatment of a subject having adisease or condition associated with apoptosis, which comprisesadministering an effective amount of a 15-keto-prostaglandin compoundrepresented by the following formula (I):

wherein W₁, W₂ and W₃ are carbon or oxygen atoms; L, M and N arehydrogen, hydroxy, halogen, lower alkyl, lower alkoxy,hydroxy(lower)alkyl or oxo, wherein at least one of L and M is a groupother than hydrogen, and the five-membered ring may have one or moredouble bond(s); A is —CH₂OH, —COCH₂OH, —COOH or its functionalderivative; B is —CH₂—CH₂—, —CH═CH— or —C≡C—; R₁ is a divalent saturatedor unsaturated lower-medium aliphatic hydrocarbon residue, which isunsubstituted or substituted by halogen, alkyl, hydroxy, oxo, aryl orheterocyclic group; and Ra is a saturated or unsaturated lower-mediumaliphatic hydrocarbon residue, which is unsubstituted or substituted byhalogen, oxo, hydroxy, lower alkyl, lower alkoxy, lower alkanoyloxy,cyclo(lower)alkyl, cyclo(lower)alkyloxy, aryl, aryloxy, heterocyclicgroup or heterocyclic-oxy group; cyclo(lower)alkyl;cyclo(lower)alkyloxy; aryl; aryloxy; heterocyclic group; orheterocyclic-oxy group to the subject.
 2. The method of claim 1, whereinthe 15-keto-prostaglandin compound is a13,14-dihydro-15-keto-prostaglandin compound.
 3. The method of claim 1,wherein the 15-keto-prostaglandin compound is a 15-keto-16-mono ordihalogen-prostaglandin compound.
 4. The method of claim 1, wherein the15-keto-prostaglandin compound is a 13,14-dihydro-15-keto-16-mono ordi-halogen-prostaglandin compound.
 5. The method of claim 1, wherein the15-keto-prostaglandin compound is a 15-keto-16-mono ordi-fluoro-prostaglandin compound.
 6. The method of claim 1, wherein the15-keto-prostaglandin compound is a 13,14-dihydro-15-keto-16-mono ordi-fluoro-prostaglandin compound.
 7. The method of claim 1, wherein the15-keto-prostaglandin compound is a 15-keto-20-lower alkyl-prostaglandincompound.
 8. The method of claim 1, wherein the 15-keto-prostaglandincompound is a 15-keto-20-ethyl-prostaglandin compound.
 9. The method ofclaim 1, wherein the 15-keto-prostaglandin compound is a2-decarboxy-2-(2-carboxy lower alkyl)-15-keto-prostaglandin compound.10. The method of claim 1, wherein the 15-keto-prostaglandin compound isa 2-decarboxy-2-(2-carboxyethyl)-15-keto-prostaglandin compound.
 11. Themethod of claim 1, wherein the 15-keto-prostaglandin compound is a2-decarboxy-2-(2-carboxyethyl)-13,14-dihydro-15-keto-16-mono ordi-fluoro prostaglandin compound.
 12. The method of claim 1, wherein the15-keto-prostaglandin compound is a2-decarboxy-2-(2-carboxyethyl)-13,14-dihydro-15-keto-16-mono ordi-fluoro-20-ethyl-prostaglandin compound.
 13. The method of claim 1,wherein the 15-keto-prostaglandin compound is a2-decarboxy-2-(2-carboxyethyl)-13,14-dihydro-15-keto-16,16-difluoro-20-ethyl-prostaglandincompound.
 14. The method of claim 1, wherein the 15-keto-prostaglandincompound is a 15-keto-prostaglandin E compound.
 15. The method of claim1, wherein the 15-keto-prostaglandin compound is2-decarboxy-2-(2-carboxyethyl)-13,14-dihydro-15-keto-16,16-difluoro-20-ethyl-prostaglandinE₁ isopropyl ester.
 16. The method of claim 1, wherein the disease orcondition associated with apoptosis is an eye disorder caused by light.17. The method of claim 1, which comprises administering ophthalmicallya composition comprising a 15-keto-prostaglandin compound formulated ina dosage form suitable for ophthalmic administration.
 18. The method ofclaim 17, wherein said composition is formulated as eye drops.